A fluorescent derivative (DTG) of thapsigargin (TG) was synthesized by replacing the C8-butanoyl chain with a dansyl moiety. Its structure was confirmed by NMR spectroscopy. Functional studies indicated that DTG retains the inhibitory effect on TG on the sarcoplasmic reticulum (SR) ATPase, displaying a 2 fM KI. Steady-state fluorescence measurements were consistent with energy transfer to DTG from tryptophanyl residues assigned to the ATPase membrane bound region. This phenomenon exhibited saturation behavior, occurred in the presence of DTG concentrations producing ATPase inhibition, and was prevented by inhibitory concentrations of TG. No energy transfer between DTG and a fluorescein 5~-isothiocyanate (FITC) label of the ATPase extramembranous region was detected. These observations suggest the inhibitory TG binding domain residues within or near the membrane bound region of the ATPase.